Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.     

Lipid transport in microorganisms

Summary Microorganisms are useful model systems for the study of intracellular transport of lipids. Eukaryotic microorganisms, such as the yeastSaccharomyces cerevisiae, are similar to higher eukaryotes with respect to organelle structure and membrane assembly. Experiments in vivo showed that transport of phosphatidylcholine between yeast microsomes and mitochondria is energy independent; transfer of phosphatidylinositol to the plasma membrane and the flux of secretory vesicles take place by different mechanisms. Linkage of transfer and biosynthesis of phospholipids was demonstrated in the case of intramitochondrial phospholipid transfer. A yeast phosphatidylinositol/phosphatidylcholine transfer protein, which is essential for cell viability, was isolated and characterized. Another phospholipid transfer protein present in yeast cytosol, which has a different specificity, is currently under investigation. Transfer of phospholipids between cellular membranes was also demonstrated with prokaryotes. The cytoplasm and the periplasma of the gram-negative facultative photosynthetic bacteriumRhodopseudomonas sphaeroides contain phospholipid transfer proteins; these seem to be involved in the biosynthesis of prokaryotic membranes.     

Lipid transfer in plants

Summary Plant cells contain cytosolic proteins, called lipid transfer proteins (LTP), which are able to facilitate in vitro intermembrane transfer of phospholipids. Proteins of this kind from three plants, purified to homogeneity, have several properties in common: molecular mass around 9 kDa, high isoelectric point, lack of specificity for phospholipids, and binding ability for fatty acids. The comparison of their amino acid sequences revealed striking homologies and conserved domains which are probably involved in their function as LTPs. These proteins could play a major role in membrane biogenesis by conveying phospholipids from their site of biosynthesis to membranes unable to form these lipids. Immunochemical methods were used to establish an in vivo correlation between membrane biogenesis and the level of LTP or the amount of LTP synthesized in vitro from mRNAs. The recent isolation of a full-length cDNA allows novel approaches to studying the participation of LTPs in the biogenesis of plant cell membranes.     

Lipid interactions with transmembrane proteins

Magnetic resonance results, principally from ²H-nuclear magnetic resonance, indicate that the mean lipid-chain ordering at the surface of transmembrane proteins is comparable to that in fluid lipid bilayers. Principally, it is the requirement for matching the hydrophobic lengths of lipid and protein that modulates the degree of chain ordering at the lipid-protein interface. The distribution of chain order parameters is, nonetheless, broader in the presence of integral proteins than in fluid lipid bilayers. The chain configurations of the phospholipids that are resolved in crystals of integral membrane proteins display considerable conformational heterogeneity. Chain C–C dihedral angles are, however, not restricted to the energetically allowable trans and gauche rotamers. This indicates that the chains of a given lipid do not have a unique configuration in protein crystals.     

Claudins: multifunctional players in epithelial tight junctions and their role in cancer

The molecular architecture of tight junctions has been a subject of extensive studies that have shown tight junctions to be composed of many peripheral and integral membrane proteins. Claudins have been considered the main tight junction-forming proteins; however, the role they play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Increasing evidence from in vitro and in vivo studies have identified the influence of claudins on tight junction structure and function, although claudins also participate in cellular contexts other than tight junctions. The aim of this review is to summarize and discuss the conceptual framework concerning claudins, focusing on the involvement of these proteins in epithelial cell polarity establishment, paracellular transport control, signal transduction and tumorigenesis. Received 5 July 2006; received after revision 29 August 2006; accepted 29 September 2006     

Transmembrane movements of lipids

Summary Membranes allow the rapid passage of uncharged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

Transmembrane movements of lipids

Membranes allow the rapid passage of unchanged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

Regulation of SNARE fusion machinery by fatty acids

Vesicle fusion is a ubiquitous biological process involved in membrane trafficking and a variety of specialised events such as exocytosis and neurite outgrowth. The energy to drive biological membrane fusion is provided by fusion proteins called SNAREs. Indeed, SNARE proteins play critical roles in neuronal development as well as neurotransmitter and hormone release. SNARE proteins form a very tight alpha-helical bundle that can pull two membranes together, thereby initiating fusion. Whereas a great deal of attention has been paid to partner proteins that can affect SNARE function, recent genetic and biochemical evidence suggests that local lipid environment may be as important in SNARE regulation. Direct lipid modification of SNARE fusion proteins and their regulation by fatty acids following phospholipase action will be discussed here in detail. Our analysis highlights the fact that lipids are not a passive platform in vesicle fusion but intimately regulate SNARE function. Received 20 December 2006; received after revision 6 February 2007; accepted 15 March 2007     

DiC14-amidine confers new anti-inflammatory properties to phospholipids

The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways.This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments. Received 12 November 2007; received after revision 4 December 2007; accepted 4 December 2007     

Thiyl radicals in biosystems: effects on lipid structures and metabolisms

Thiyl radicals are intermediates of enzyme- and radical-driven biochemical processes, and their potential as reactive species in the biological environment has been somehow underestimated. From organic chemistry, however, it is known that thiyl radicals isomerize the double bonds of unsaturated fatty acids to a mixture with very dominating trans isomers. Recently, this reaction has been particularly studied for biosystems, focusing on the effect of thiyl radicals on the natural all-cis double bonds of unsaturated phospholipids, which undergo a conversion to the unnatural trans form. In this paper we report briefly the role of thiyl radicals in biosystems, describe the main features of the radical-induced cis-trans isomerization process under both in vitro and in vivo conditions, and reflect on some consequences for membrane structures, lipid metabolism and enzymatic reactions.Received 29 October 2004; received after revision 3 December 2004; accepted 4 January 2005     

The function of apolipoproteins L

The function of the proteins of the apolipoprotein L (apoL) family is largely unknown. These proteins are classically thought to be involved in lipid transport and metabolism, mainly due to the initial discovery that a secreted member of the family, apoL-I, is associated with high-density lipoprotein particles. However, the other members of the family are believed to be intracellular. The recent unravelling of the mechanism by which apoL-I kills African trypanosomes, as well as the increasing evidence for modulation of apoL expression in various pathological processes, provides new insights about the functions of these proteins. ApoLs share structural and functional similarities with proteins of the Bcl-2 family. Based on the activity of apoL-I in trypanosomes and the comparison with Bcl-2 proteins, we propose that apoLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death. Received 28 February 2006; received after revision 18 May 2006; accepted 2 June 2006     

Polar transport of the plant hormone auxin – the role of PIN-FORMED (PIN) proteins

The PIN-FORMED (PIN) protein family is a group of plant transmembrane proteins with a predicted function as secondary transporters. PINs have been shown to play a rate-limiting role in the catalysis of efflux of the plant growth regulator auxin from cells, and their asymmetrical cellular localization determines the direction of cell-to-cell auxin flow. There is a functional redundancy of PINs and their biochemical activity is regulated at many levels. PINs constitute a flexible network underlying the directional auxin flux (polar auxin transport) which provides cells in any part of the plant body with particular positional and temporal information. Thus, the PIN network, together with downstream auxin signalling system(s), coordinates plant development. This review summarizes recent progress in the elucidation of the role of PIN proteins in polar auxin transport at the cellular level, with emphasis on their structure and evolution and regulation of their function. Received 28 December 2006; received after revision 16 February 2007; accepted 26 March 2007     

Bridging the molecular and biological functions of the oxysterol-binding protein family

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.     

Apical protein transport

The plasma membrane of epithelial cells and hepatocytes is divided into two separate membrane compartments, the apical and the basolateral domain. This polarity is maintained by intracellular machinery that directs newly synthesized material into the correct target membrane. Apical protein sorting and trafficking require specific signals and different intracellular routes to the cell surface. Some of them depend on the integrity of sphingolipid/cholesterol-enriched membrane microdomains named ‘lipid rafts’, others use separate transport platforms. Certain characteristics of the heterogeneous population of apical sorting signals are described in this review and cellular factors associated with sorting and transport mechanisms are discussed. Received 5 May 2006; received after revision 12 June 2006; accepted 11 July 2006     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

The assembly of lipids into lipoproteins during secretion

Summary The process of assembly and secretion of lipoproteins is discussed with particular reference to the role of lipids. The majority of circulating lipoproteins is produced by the liver (80%) with the remainder being supplied by the intestine. The liver secretes both very low density lipoproteins and high density lipoproteins, but the assembly and secretion of these two types of particles may follow different routes. The major lipid components of lipoproteins are triacylglycerols, cholesterol, cholesterol esters and phospholipids. The biosynthesis of these lipids occurs on membranes of the endoplasmic reticulum, with many of the enzymes also being present in the Golgi; the roles of these two subcellular organelles in the assembly of lipoproteins are discussed. There appears to be a compartmentalization of lipids in cells, such that defined pools, often those newly-synthesized, are preferred, or even required, for lipoprotein assembly. The process of hepatic very low density lipoprotein secretion appears to be regulated by the supply of lipids. Indeed, the synthesis of new lipid may be a major driving force in lipoprotein assembly and secretion.     

The assembly of lipids into lipoproteins during secretion

The process of assembly and secretion of lipoproteins is discussed with particular reference to the role of lipids. The majority of circulating lipoproteins is produced by the liver (80%) with the remainder being supplied by the intestine. The liver secretes both very low density lipoproteins and high density lipoproteins, but the assembly and secretion of these two types of particles may follow different routes. The major lipid components of lipoproteins are triacylglycerols, cholesterol, cholesterol esters and phospholipids. The biosynthesis of these lipids occurs on membranes of the endoplasmic reticulum, with many of the enzymes also being present in the Golgi; the roles of these two subcellular organelles in the assembly of lipoproteins are discussed. There appears to be a compartmentalization of lipids in cells, such that defined pools, often those newly-synthesized, are preferred, or even required, for lipoprotein assembly. The process of hepatic very low density lipoprotein secretion appears to be regulated by the supply of lipids. Indeed, the synthesis of new lipid may be a major driving force in lipoprotein assembly and secretion.     

Regulation of membrane trafficking and endocytosis by protein kinase C: emerging role of the pericentrion,a novel protein kinase C-dependent subset of recycling endosomes

The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization. Received 11 August 2006; received after revision 20 September 2006; accepted 7 November 2006     

Bardet-Biedl syndrome: an emerging pathomechanism of intracellular transport

From a handful of uncloned genetic loci 6 years ago, great strides have been made in understanding the genetic and molecular aetiology of Bardet-Biedl syndrome (BBS), a rare pleiotropic disorder characterised by a multitude of symptoms, including obesity, retinal degeneration and cystic kidneys. Presently, 11 BBS genes have been cloned, with the likelihood that yet more BBS genes remain undiscovered. In 2003, a major breakthrough was made when it was shown that BBS is likely caused by defects in basal bodies and/or primary cilia. Since then, studies in numerous animal models of BBS have corroborated the initial findings and, in addition, have further refined the specific functions of BBS proteins. These include roles in establishing planar cell polarity (noncanonical Wnt signaling) in mice and zebrafish, modulating intraflagellar transport and lipid homeostasis in worms, and regulating intracellular trafficking and centrosomal functions in zebrafish and human tissue culture cells. From these discoveries, a common theme has emerged, namely that the primary function of BBS proteins may be to mediate and regulate microtubule-based intracellular transport processes. Received 20 April 2006; received after revision 30 May 2006; accepted 15 June 2006